US 7,585,636 B2
Protein subcellular localization assays using split fluorescent proteins
Geoffrey S. Waldo, Santa Fe, N. Mex. (US); and Stephanie Cabantous, Los Alamos, N. Mex. (US)
Assigned to Los Alamos National Security, LLC, Los Alamos, N. Mex. (US)
Filed on Dec. 05, 2005, as Appl. No. 11/295,374.
Claims priority of provisional application 60/633488, filed on Dec. 04, 2004.
Prior Publication US 2006/0257942 A1, Nov. 16, 2006
Int. Cl. G01N 33/567 (2006.01); G01N 33/53 (2006.01); C12Q 1/68 (2006.01)
U.S. Cl. 435—7.1  [435/6; 435/7.2; 435/320.1; 435/325; 435/455; 435/69.1; 530/350; 536/23.5] 14 Claims
 
1. An assay for detecting the localization of a test protein, X, to a subcellular component of interest in a cell, comprising:
(a) expressing in the cell or providing to the cell (i) a fusion protein comprising the test protein, X, and a microdomain tag fragment of a fluorescent protein, wherein the microdomain tag fragment corresponds to one or more contiguous beta-strands of the fluorescent protein, and (ii) a complementary assay fragment of the fluorescent protein functionalized to be directed and localized to the subcellular component of interest, wherein the assay fragment corresponds to the remaining contiguous beta-strands of the fluorescent protein, and wherein the microdomain tag and complementary assay fragments self-complement to reconstitute the fluorescent protein if present in the same subcellular component; and,
(b) detecting fluorescence in the cell, and thereby detecting the localization of the test protein, X, to the subcellular component of interest.